Acetylisovaleryltylosin tartrate (ATLL) is a new macrolide veterinary antibiotic, it is necessary to study the binding of ATLL to protein, which will directly correlate with the efficiency in vivo. Bovine serum albumin (BSA) is structure homologous with human serum albumin (HSA), and is commonly chosen as a model to investigate drug-protein interaction.There are many metal ions in plasma, as yet, the studies on mainly focus on single metal ion. In this study, the multiple systems formed by ATLL and BSA without or with Zn2+ and Cu2+ have been studied by mult-spectroscopy. The results showed that, the fluorescence of BSA was quenched by ATLL through a static quenching mechanism. The effective quenching constant (Ka) of ATLL to BSA decreased with Zn2+ and increased with Cu2+. Thermodynamic parameters revealed that hydrogen bonds and hydrophobic forces played significant roles in the binding of ATLL to BSA. The polarity of tryptophan and tyrosine residues changed when adding ATLL with or without Zn2+ and Cu2+. FT-IR spectra showed that ATLL changed α-helix and β-sheet of BSA into β-turn and random structure. The UV-Vis spectra indicated that the effects of Zn2+ on ATLL binding to BSA may cause by a competition binding, and Cu2+ possibly formed Cu2+-ATLL complex via metal ion bridge. All the knowledge obtained in this work will be helpful to understand the transport mechanism of ATLL with BSA and the effect of metal ions on the interaction of drug-protein in vivo.